The relationship between α/β and γ/δ T-cell lineages was studied in rats using RT-PCR analysis of TCRβ transcripts in γ/δ T-cell hybridomas and an intracellular staining technique to detect TCRβ protein in primary γ/δ T-cells. We report the presence of functional TCRβ transcripts in 2/9 γ/δ T-cell hybridomas. About 15 % of peripheral γ/δ T-cells and thymocytes also express TCRβ protein, giving a minimum estimate for successful Tcrb rearrangement based on exvivo single cell analysis. In athymic rats, γ/δ T-cells expressing intracellular β protein are present but at a lower frequency than in euthymic controls, suggesting that in the thymus, more γ/δ T-cell precursors pass through a stage where functional β rearrangement has occurred than in extrathymic sites. Analysis of TCR expression in purified transitory immature CD4-8+ (iCD8SP) thymocytes and their spontaneously developing CD4+8+ (DP) progeny showed that TCRγ mRNA is expressed in iCD8SP cells but not in their immediate DP progeny that reinitiate RAG-1 transcription and commence α/βTCR expression. We conclude that rat γ/δ T cells can separate from the α/β lineage after TCRβ expression, but not after entry into the DP compartment.